Hereditary orotic aciduria—Pyrimidine auxotrophism in man

The American Journal of Medicine JANUARY

Vol. 38

1965

Editorial Hereditary

Orotic Aciduria-Pyrimidine

Auxotrophism I

H

in Man*

erotic acid to uridine-5’-phosphate [5]. (Fig. 1.) Fetal development appears to be normal, but anemia, leukopenia, increased susceptibility to infection and retarded growth and development imperil neonatal survival. Uridine therapy results in prompt reversal of the chemical, cytological and clinical manifestations of the disease, a response sustained only during the period of continued treatment [2,3]. (Fig. 2.) A cell line of fibroblasts from a patient with hereditary erotic aciduria requires pyrimidine nucleoside supplementation in the tissue culture medium for optimal growth [6]. Hereditary erotic aciduria is, therefore, pyrimidine auxotrophism in man. There is no precise analogy in other known human genetic disorders for this kind of acquired auxotrophism for a biosynthetic intermediate. Thyroxine in familial cretinism, a glucocorticoid in severe forms of congenital adrenal virilism, immune globulins in congenital agammaglobulinemia, or antihemophihc globulin in a bleeding crisis of hemophilia A may be required in their special homeostatic functions for survival. This is replacement treatment with the circulating end product of a genetically impaired synthesis. One may inquire why true auxotrophism (purine, pyrimidine, porphyrin

erotic aciduria is a rare genetic disorder of pyrimidine metabolism characterized by megaloblastic anemia unresponsive to usual hematinic therapy, retarded growth and development, and excessive urinary excretion of erotic acid [I$‘]. Only three patients with this condition have been discovered [1,3/I]. There are several features of the disease which lend added importance to its investigation: (1) it is the only genetic disorder of pyrimidine or purine nucleotide synthesis so far elucidated in man or in any nonmicrobial organism; (2) it can be partially simulated by use of a pharmacological agent; (3) it represents “ pyrimidine starvation” in man, illustrating intracellular metabolic control mechanisms previously described in microorganisms ; and (4) the presence of a double enzyme defect suggests a mutation in a regulatory gene, i.e., an operon disorder. Pyrimidine Auxotrofihism. Pyrimidine auxotrophs are microbiological mutants with genetic deletions of enzymes in the de nova pathway of pyrimidine biosynthesis. A preformed pyrimidine becomes a nutritional requirement for growth. Hereditary erotic aciduria is unique as an “involuted genetic disorder” in man in that it is a genetic defect in the production of genetic material. There is a block in the conversion of EREDITARY

* This work was supported by Grant A.M. 5846, National Institute of Arthritis and Metabolic Disease.

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FIG. 2. Response of a patient with hereditary erotic aciduria during treatment with uridine [3].

or creatine for example) is not more common in human disease, since these biosynthetic pathways represent large numbers of specific enzymes under mutation hazard. A homozygous defect in such a major pathway must ordinarily be lethal. In microorganisms, auxotrophic mutants are discovered by their growth require-

ments in culture media. In man the auxotrophic . zygote cannot obtam from intrauterine sources the necessary metabolite for survival. Hereditary erotic aciduria is transmitted as an autosomal trait [7]. Heterozygotes have reduced activities of orotidylic pyrophosphorylase and orotidylic decarboxylase in their erythroAMERICAN

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Editorial cytes [Z], leukocytes [B] and cell lines of fibroblasts [6], and excrete a small excess of erotic acid in their urine [9]. They have no hematologic or clinical abnormalities. Twenty-two heterozygotes have been found in the families of the three patients and an additional presumed heterozygote has turned up in a small control series [Z]. The incidence of this mutant gene has not been established. Homozygous hereditary erotic aciduria is a very rare disease. It is not clear how these few children underwent normal embryological development despite a genetic block in a pathway required for the synthesis of DNA, RNA and essential pyrimidine nucleotide cofactors. There are several possible explanations. In theory de novo synthesis of pyrimidines might occur by an alternate pathway exclusive of erotic acid, which would presumably undergo a compensatory increase in activity. As yet no alternate pathway has been discovered in mammalian systems. Pyrimidine nucleotides might be formed by salvage synthesis, a term for the reutilization of preformed pyrimidine bases available from nucleotide turnover or from dietary sources. In fetal development, the original pyrimidine ring would derive from maternal synthesis. Plasma levels of pyrimidines, however, are low, and uracil treatment was ineffective in the first described patient [I]. In a cell line of fibroblasts from a patient with hereditary erotic aciduria, activity of the affected enzymes was not wholly absent, but approximated 0.4 to 2.0 per cent of the normal level [6]. The residual activity may represent that of a structurally altered enzyme, a regulatory gene defect which reduces but does not delete structural gene expression, or interallelic complementation [ 701. Isoenzymes under different genetic control may be present in tissues other than those so far tested. The marginal survival of patients with hereditary erotic aciduria must depend on some such form of genetic leakage. Drug-Induced Orotic Aciduria. A disorder of pyrimidine metabolism somewhat analogous to genetically-induced erotic aciduria is produced in man by the antineoplastic agent 6azauridine [ 7 7 1. Following its conversion to 6azauridylic acid, this compound is a specific competitive inhibitor of orotidylic decarboxylase [72], an effect which can be demonstrated as reduced decarboxylation of erotic acid in viuo [73]. Its action may be more complex since 6azauracil has been shown to disrupt the coordination of pyrimidine enzyme activities in EscherVOL.

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JANUARY

1965

3

ichia coli [74]. Furthermore over-all synthesis of pyrimidines, estimated by indirect methods, may not be reduced during 6-azauridine administration in man [ 751. Orotic acid and orotidine (Fig. 1) are present in normal urine in amounts of approximately 1.5 and 2.5 mg., respectively [9]. 6-Azauridine leads to a prompt 5,000- to 8,000-fold increase in their excretion, which disappears quickly when the drug is withdrawn. Prolonged use of 6azauridine may lead to anemia and megaloblastosis, similar to but less severe than that of the genetic disorder [76]. Megaloblastosis is thought to reflect unbalanced growth of the cell with selective impairment of DNA synthesis [ 771. Its occurrence in hereditary erotic aciduria is provocative in that the enzymatic defect lies on a pathway common to both DN.4 and RNA synthesis. Orotic acid excretion in the hereditary disorder approximates that induced by 6-azauridine, when corrections are made for body size. In contrast, the orotidine in the urine is increased only ten- to twentyfold, a level of orotidinuria less than 1 per cent that of the druginduced disorder [ 781. This major difference is presumably the result of the double defect in hereditary erotic aciduria involving both enzymes which catalyze the conversion of erotic acid to uridine-5’-phosphate. (Fig. 1.) 6-Azauridine is but one example of a drug which simulates by enzyme inhibition a genetic disorder of enzyme deletion. Other examples are methopyrapone inhibition of adrenocortical 1 la-hydroxylase simulating the hypertensive form of congenital adrenal virilism [79] and 4hydroxypyrazolopyrimidine inhibition of xanthine oxidase simulating xanthinuria [ZO]. Release of Regulatory Mechanisms. Normal synthesis of pyrimidines in man approximates 0.6 gm. per 24 hours, an amount roughly equimolar with purine synthesis [27]. In hereditary erotic aciduria, excretion of erotic acid (eorrected for body size) may exceed this figure more than tenfold. The degree of erotic aciduria is more immediately the result of overproduction than underutilization. There are two general types of intracellular regulatory mechanisms: (1) regulation of the rate of synthesis of enzymes, and (2) regulation of enzyme activity (probably through structural modifications). Examples of enzyme induction and repression are still rare in mammalian systerns, and the changes in enzyme levels are less

Editorial in vivo. Uridine administration alleviates pyrimidine starvation and, by renewal of end product repression and feedback inhibition, reduces erotic acid excretion. (Fig. 2.) The effect of glucocorticoid treatment on the hormonal imbalance of congenital adrenal hyperplasia may be considered as a specialized analogy. This general pattern of metabolic regulation of a biosynthetic pathway is perhaps more clearly illustrated in hereditary erotic aciduria than in any other genetic disease. Double Enzyme Defect. Hereditary erotic aciduria is the only genetic disease so far elucidated in which two adjacent enzymes have been involved. There are several possible interpretations of such a double enzyme defect. Are both biochemical steps catalyzed by the same enzyme? The two enzymatic activities have been separated by purification procedures in yeast and rat liver preparations. They can be selectively inhibited by pyrimidine analogues and exhibit different stabilities on storage. Separation of orotidylic pyrophosphorylase and orotidylic decarboxylase activities has not yet been carried out in human material, but it seems unlikely that they represent two active sites on a single protein. Does a genetic defect of one enzyme lead to a secondary nongenetic loss of activity of the adjacent enzyme? This is more difficult to exclude in man. In Esch. coli mutants with a selective block at either orotidylic pyrophosphorylase or orotidylic decarboxylase, growth in a pyrimidine-limited medium leads to an increase rather than a decrease in activity of the other enzyme 1271. This would be anticipated from release of repression during pyrimidine starvation. In man, selective inhibition of orotidylic decarboxylase by 6-azauridylic acid produces orotidinuria as well as erotic aciduria. This presumably reflects increased rather than reduced activity of orotidylic pyrophosphorylase. Although these analogies are not conclusive, they suggest by exclusion a genotypic origin for the double enzyme defect of hereditary erotic aciduria. Is there a coincidental mutation of two structural genes? In Esch. coli the loci which code the structure of four sequential enzymes in pyrimidine biosynthesis-dihydroorotase, dihydroorotic dehydrogenase, orotidylic pyrophosphorylase and orotidylic decarboxylase-are closely linked [74]. If similar linkage occurs on the human chromosome, a single extensive defect might involve the structural gene coding sites for two derepression

ENZYMES FIQ. 3. Scheme for the genetic control of enzyme synthesis, as proposed by Jacob and Monod [Z8].

marked than those in bacteria [22]. Uracil represses the synthesis of enzymes which catalyze the de novo biosynthetic pathway in pyrimidinerequiring Esch. coli mutants. Growth of these mutants in media limited in pyrimidine supplementation leads to an increase in the synthesis of enzymes prior to the site of the genetic block. Thii is the pattern of “pyrimidine starvation,” mediated by release of endproduct repression of enzyme formation [23]. Repression and release of repression, or derepression, are general regulatory mechanisms for accommodating enzyme levels to metabolic needs. In addition to enzyme synthesis, enzyme activity may be altered. These changes in enzyme activity probably result from structural modifications of the molecule-association-dissociation into subunits, reversible inactivation, or more subtle conformational changes produced by binding of an inhibitory agent at an allosteric site [24]. Feedback inhibition of the first step unique to a biosynthetic sequence by one of the end products is a widely recognized example of this type of control mechanism. Cytidine triphosphate competitively inhibits aspartate transcarbamylase, the first enzyme unique to pyrimidine biosynthesis, presumably by allosteric binding [25]. Other product inhibitions of enzyme activity may occur within the pyrimidine sequence. Hereditary erotic aciduria is pyrimidine starvation in man. Earlier enzymes in pyrimidine synthesis (aspartate transcarbamylase and dihydroorotase) are present in increased activity in erythrocytes, probably as a result of derepression [ZSJ. Release of negative feedback inhibition presumably contributes to excessive erotic acid synthesis, although this effect on enzyme activity cannot be easily differentiated from

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Editorial enzymes. (Fig. 3.) The presence of a double enzyme defect in all families studied makes such a fortuitouslv located chromosomal lesion improbable. However, this possibility cannot be excluded. Is there a mutation in a genetic regulatory mechanism which controls the synthesis of two enzymes? Studies of the genetic control of enzyme formation in microorganisms has led to the hypothesis represented in Figure 3 [28]. A cluster of structural genes may be under the control of an adjacent operator gene, constituting a genetic unit of transcription termed an operon. The activity of the operator gene, and therefore the coordinated expression oi all structural genes within the operon, is in turn under the control of a regulator gene. This control is exercised through the synthesis of a repressor substance. The operon hypothesis has not been established as applicable in mammalian cells. The presence of a double enzyme disorder in hereditary erotic aciduria is consistent with a defect of an operator or a regulator gene. This implies that the genes which code the structure of orotidylic pyrophosphorylase and orotidylic decarboxylase, together with an adjacent operator gene, constitute an operon on the human chromosome. This speculation that hereditary erotic aciduria may represent an operon disorder in man remains to be established.

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LLOYD H. SMITH, JR.

University of California San Francisco Medical Center San Francisco, California

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18. REFERENCES 19. 1. HUGULEY, C. M., JR., BAIN, J. A., RIVERS, S. and SCOGGINS,R. Refractory megaloblastic anemia associated with excretion of erotic acid. Blood, 14: 615,1959. 2. SMITH, L. H., JR., HUGULEY, C. M., JR. and BAIN, J. A. Hereditary erotic aciduria In: The Metabolic Basis of Inherited Disease, 2nd ed. Edited by Stanbury, J. B., Wyngaarden, J. B. and Frederickson, D. S., Neiv York, McGraw-Hill Book Co., in press. 3. BECROFT, D. M. 0. and PHILLIPS, L. I. Hereditary erotic aciduria with megaloblastic anaemia. A second case with response to uridine. In preparation. 4. HAGGARD, M. E. Unpublished observations. 5. SMITH, L. H., JR., SULLIVAN, M. and HUGULEY, C. M., JR. Pyrimidine metabolism in man. IV. The enzymatic defect of erotic aciduria. J. Clin. Invest., 40: 656, 1961. VOL.

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K~oort~. R. S., HOWELL, R. R. and KLINENH~RG. J. A. Properties of diploid cell strains developed from patients with an inherited ahnormality of uridine biosynthesis. Cold .Yprinq flurbor Syrnfi. Quant. Biol., in press. FALLON, H. J., SMITH, L. H., .JR., GRAHM, J. B. and BURNETT,C. H. A genetic study of hrreditary orotic aciduria. Neru England J. L21ed.>270 : 878, 1964. FALLON, H. J., LOTZ, M. and SMITH. L. I-I., JR. Congenital erotic aciduria: demonstration of an enyzme defect in leukocytes and comparison with drug-induced erotic aciduria. Blood, 20 : 700, 1962. LOTZ, M., FALLON, H. J. and SMITII. L. H., JR. Excretion of erotic acid and orotidine in heterozygotes of congenital erotic aciduria. ,Vuture,197: 194,1963. FINCHAM,J. R. S. and CODDINGTON,A. Complementation at the am locus of Neueurosporn crassa: a reaction between different mutant forms of glutamic dehydrogenase. J. M&c. Biol., 6: 361, 1963. CARDOSO, S. S., CALABRESI, P. and HANDSCHUMACIIER,R. E. Alterations in human pyrimidine metabolism as a result of therapy with 6-azauridine. Cancer Res., 21: 1551, 1961. HANDSCIIUMACHER, R. E. Orotidylic acid decarboxylase. Inhibition studies with azauridine 5 ‘-phosphate. J. Biol. Chem., 235: 2917, 1960. RABKIN, M. T., FREDERICK,E. W., LOTZ, M. and SMITH, L. H., JR. Pyrimidine metabolism in man. v. The measurement in z&o of the biochemical effect of antineoplastic agents in animal and human subjects. J. Clin. Invest., 41 : 871, 1962. BECKWITH, J. R., PARDEE, A. B., AUSTRIAN,R. and JACOB, F. Co-ordination of the synthesis of the enzymes in the pyrimidine pathway of E. coli. J. M&c. Biol., 5: 618, 1962. BONO, V. H., JR., WEISSMAN,S. M. and FREI, E., III. The effect of 6-azauridine administration on de nova pyrimidine production in chronic myelogenous leukemia. J. Clin. Invest., 43: 1486, 1964. CALABRESI,P. Personal communication. REISNER, E. H., JR. The nature and significance of megaloblastic blood formation. Blood, 13: 313, 1958. BAIN, J. A. and HUGULEY, C. M., JR. Unpublished observations. LIDDLE, G. W., ISLAND,D., LANCE, E. M. and HARRIS, A. P. Alterations of adrenal steroid patterns in man resulting from treatment with a chemical inhibitor of 11 P-hydroxylation. J. Clin. Endocrinol., 18: 906, 1958. RUNDLES, R. W., WYNGAARDEN, J. B., HITCHINGS, G. I-I., ELION, G. B. and SILBERMAN,H. R. Effects ofaxanthineoxidaseinhibitoron thiopurine metabolism, hyperuricemia, and gout. Tr. A. .4m. Physicians, 76: 126, 1963. WEISSMAN,S. M., EISEN, A. Z., FALLON, H., LEWIS, M. and KARON, M. The metabolism of ring-labeled erotic acid in man. J. Clin. Znwst.. 41 : 1546, 1962. PARDEE,A. B. and WILSON, A. C. Control of enzyme activity in higher animals. Cancer Ra., 23: 1483, 1963. YATES, R. A. and PARDEE, A. B. Control by uracil of formation of enzymes required for orotate synthesis. J. Biol. Chem., 227 : 677, 1957.

Editorial 24. MONOD, J., CHANGEUX, J.-P. and JACOB, F. Allosteric proteins and cellular control systems. J. Molec. Biot., 6: 306,1963. 25. GERHART, J. C. and PARDEE, A. B. The enzymology of control by feedback inhibition. J. Biol. Chem., 237: 891,1962. 26. SETH, L. H., JR. Unpublished observations.

27. SMITH, L. H., JR. and LOTZ, M. Studies on congenital erotic aciduria: comparison of erotic acid metabolism in microorganisms. J. Lab. tY Clin. Med., 61: 211,1963. 28. JACOB, F. and MONOD, J. Genetic regulatory mechanisms in the synthesis of proteins. J. Molec. Biol., 3: 318, 1961.

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